This project involves the application of the techniques of molecular biology to a basic problem in channel biophysics: What are the molecular mechanisms that underlie the functioning of ion channels? This study will focus on the nicotinic acetylcholine receptor (AChR), which is by far the best-characterized channel from an electrophysiological, biochemical, and structural aspects. Site-directed mutagenesis will be used to introduce mutations in a cDNA that codes forone of the receptor subunits. The mutations are chosen to change a single amino acid in a predetermined position. Mutant and wild-type cDNAs are used to produce large quantities of mRNA by in vitro transcription and the appropriate mixture of mRNAs is injected into Xenopus oocytes, which then produce functional AChRs. These mutant receptors are studied by a combination of electrophysiological and biochemical techniques. Comparison of the effects of various mutations to the properties of the wild-type AChRs will enable us to map out the functionally relevant structural features, such as the ligand-binding site and the ion channel itself. It is expected that some of the structural features of the AChR will be common to other types of receptors and channels.